skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Cryo-EM structure of the human neutral amino acid transporter ASCT2

Abstract

Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for many retroviruses; hence, it has importance as a potential drug target. Here we used single-particle cryo-EM to determine a structure of the functional and unmodified human ASCT2 at 3.85-Å resolution. ASCT2 forms a homotrimeric complex in which each subunit contains a transport and a scaffold domain. Prominent extracellular extensions on the scaffold domain form the predicted docking site for retroviruses. Relative to structures of other SLC1 members, ASCT2 is in the most extreme inward-oriented state, with the transport domain largely detached from the central scaffold domain on the cytoplasmic side. In conclusion, this domain detachment may be required for substrate binding and release on the intracellular side of the membrane.

Authors:
 [1]; ORCiD logo [1];  [2]; ORCiD logo [1];  [1]; ORCiD logo [1]
  1. Univ. of Groningen, Groningen (The Netherlands)
  2. SLAC National Accelerator Lab., Menlo Park, CA (United States); Stanford Univ., Stanford, CA (United States)
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1475494
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Structural & Molecular Biology
Additional Journal Information:
Journal Volume: 25; Journal Issue: 6; Journal ID: ISSN 1545-9993
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Garaeva, Alisa A., Oostergetel, Gert T., Gati, Cornelius, Guskov, Albert, Paulino, Cristina, and Slotboom, Dirk J. Cryo-EM structure of the human neutral amino acid transporter ASCT2. United States: N. p., 2018. Web. doi:10.1038/s41594-018-0076-y.
Garaeva, Alisa A., Oostergetel, Gert T., Gati, Cornelius, Guskov, Albert, Paulino, Cristina, & Slotboom, Dirk J. Cryo-EM structure of the human neutral amino acid transporter ASCT2. United States. https://doi.org/10.1038/s41594-018-0076-y
Garaeva, Alisa A., Oostergetel, Gert T., Gati, Cornelius, Guskov, Albert, Paulino, Cristina, and Slotboom, Dirk J. Tue . "Cryo-EM structure of the human neutral amino acid transporter ASCT2". United States. https://doi.org/10.1038/s41594-018-0076-y. https://www.osti.gov/servlets/purl/1475494.
@article{osti_1475494,
title = {Cryo-EM structure of the human neutral amino acid transporter ASCT2},
author = {Garaeva, Alisa A. and Oostergetel, Gert T. and Gati, Cornelius and Guskov, Albert and Paulino, Cristina and Slotboom, Dirk J.},
abstractNote = {Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for many retroviruses; hence, it has importance as a potential drug target. Here we used single-particle cryo-EM to determine a structure of the functional and unmodified human ASCT2 at 3.85-Å resolution. ASCT2 forms a homotrimeric complex in which each subunit contains a transport and a scaffold domain. Prominent extracellular extensions on the scaffold domain form the predicted docking site for retroviruses. Relative to structures of other SLC1 members, ASCT2 is in the most extreme inward-oriented state, with the transport domain largely detached from the central scaffold domain on the cytoplasmic side. In conclusion, this domain detachment may be required for substrate binding and release on the intracellular side of the membrane.},
doi = {10.1038/s41594-018-0076-y},
url = {https://www.osti.gov/biblio/1475494}, journal = {Nature Structural & Molecular Biology},
issn = {1545-9993},
number = 6,
volume = 25,
place = {United States},
year = {2018},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 37 works
Citation information provided by
Web of Science

Figures / Tables:

Fig. 1 Fig. 1: Functional characterization of ASCT2. a, Exchange of internal unlabeled glutamine with external [ 3H]glutamine, and inhibition of transport by addition of an excess of unlabeled amino acids (circle, glutamine; diamond, tryptophan; X, no externally added amino acid (No)). b, Inhibitory effect of externally added amino acids. c, Exchangemore » of internal amino acid substrate. d, Ability of alternative internal amino acids to drive uptake of glutamine. e, Electroneutral nature of exchange and sodium dependency. Circle, 3 $$μ$$M valinomycin was added at 23 min to proteoliposomes with a 100-fold K + gradient; downward triangle, ethanol was added at 23 min to proteoliposomes with a 100-fold K + gradient; square, 3 $$μ$$M valinomycin was added at the start to proteoliposomes with a 100-fold K + gradient; upward triangle, proteoliposomes in the absence of Na +. Arrow indicates time of addition of valinomycin or ethanol (23 min). f, Exchange of internal unlabeled glutamine with external [ 3H]glutamine in proteoliposomes containing different amounts of cholesterol. Circle, proteoliposomes without cholesterol; downward triangle, with 5% (wt/wt) cholesterol; square, with 10% (wt/wt) cholesterol. Data points and error bars represent means± s.e.m. from 3 biologically independent experiments, each done in one technical replicate. Small schemes in the top left corners of a, c, e and f schematically represent proteoliposomes with internal and external compound compositions used in corresponding experiments.« less

Save / Share:

Works referenced in this record:

Transport mechanism of a bacterial homologue of glutamate transporters
journal, November 2009


Membrane transporters for the special amino acid glutamine: structure/function relationships and relevance to human health
journal, August 2014


Mechanism of Na+-dependent citrate transport from the structure of an asymmetrical CitS dimer
journal, December 2015


Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2
journal, November 2016


Expression of glutamine metabolism-related proteins according to molecular subtype of breast cancer
journal, March 2013


Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris — functional and kinetic asymmetry revealed in proteoliposomes
journal, September 2013


Glutamate uptake
journal, September 2001


Prevention of overfitting in cryo-EM structure determination
journal, July 2012


Clinical Role of ASCT2 (SLC1A5) in KRAS-Mutated Colorectal Cancer
journal, July 2017


Simian Retrovirus 4 Induces Lethal Acute Thrombocytopenia in Japanese Macaques
journal, January 2015


Voltage-dependent processes in the electroneutral amino acid exchanger ASCT2
journal, May 2013


CTFFIND4: Fast and accurate defocus estimation from electron micrographs
journal, November 2015


Structure of a glutamate transporter homologue from Pyrococcus horikoshii
journal, October 2004


Inward-facing conformation of glutamate transporters as revealed by their inverted-topology structural repeats
journal, November 2009


MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy
journal, February 2017


ASCT2/SLC1A5 controls glutamine uptake and tumour growth in triple-negative basal-like breast cancer
journal, October 2015


Analysis of the quality of crystallographic data and the limitations of structural models
journal, October 2017


ASC amino-acid transporter 2 (ASCT2) as a novel prognostic marker in non-small cell lung cancer
journal, March 2014


Structure and allosteric inhibition of excitatory amino acid transporter 1
journal, April 2017


Characterization of feline ASCT1 and ASCT2 as RD-114 virus receptor
journal, April 2013


Optimal Determination of Particle Orientation, Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy
journal, October 2003


The SLC1 high-affinity glutamate and neutral amino acid transporter family
journal, April 2013


Membrane reconstitution of ABC transporters and assays of translocator function
journal, January 2008


Coupled binding mechanism of three sodium ions and aspartate in the glutamate transporter homologue GltTk
journal, November 2016


Crystal structure of a substrate-free aspartate transporter
journal, September 2013


Neutral amino acid transporter ASCT2 displays substrate-induced Na+ exchange and a substrate-gated anion conductance
journal, March 2000


Transport domain unlocking sets the uptake rate of an aspartate transporter
journal, February 2015


MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009


Active transport of L-glutamate by membrane vesicles isolated from rat brain
journal, September 1978


Coot model-building tools for molecular graphics
journal, November 2004


Arginine 447 Plays a Pivotal Role in Substrate Interactions in a Neuronal Glutamate Transporter
journal, September 2000


Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5)
journal, July 2014


N-linked Glycosylation of human SLC1A5 (ASCT2) transporter is critical for trafficking to membrane
journal, July 2015


Cloning and Functional Characterization of a System ASC-like Na + -dependent Neutral Amino Acid Transporter
journal, June 1996


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010


Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter
journal, January 2007


Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters
journal, February 2016


Shared Molecular Mechanisms of Membrane Transporters
journal, June 2016


Flux coupling in a neuronal glutamate transporter
journal, October 1996


Structural insight in the toppling mechanism of an energy-coupling factor transporter
journal, March 2016


Targeting glutamine transport to suppress melanoma cell growth: Targeting Glutamine Transport to Suppress Melanoma Cell Growth
journal, February 2014


Structural characterisation reveals insights into substrate recognition by the glutamine transporter ASCT2/SLC1A5
journal, January 2018


Stock-based detection of protein oligomeric states in jsPISA
journal, April 2015


Focus: The interface between data collection and data processing in cryo-EM
journal, May 2017


Sampling the conformational space of the catalytic subunit of human γ-secretase
journal, December 2015


Structural Features of the Glutamate Transporter Family
journal, June 1999


    Works referencing / citing this record:

    OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics
    journal, December 2018


    Amino Acid Transporters and Exchangers from the SLC1A Family: Structure, Mechanism and Roles in Physiology and Cancer
    journal, January 2020


    Transporter oligomerisation: roles in structure and function
    journal, December 2018


    ASCT2: a potential cancer drug target
    journal, May 2019


    Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli
    journal, September 2019


    Glutamate transporters: a broad review of the most recent archaeal and human structures
    journal, August 2019


    Exploiting Cysteine Residues of SLC Membrane Transporters as Targets for Drugs
    journal, June 2019


    Binding and transport of D-aspartate by the glutamate transporter homolog GltTk
    journal, April 2019


    Models to determine the kinetic mechanisms of ion-coupled transporters
    journal, January 2019


    ASCT2: a potential cancer drug target
    journal, May 2019


    The Human SLC1A5 (ASCT2) Amino Acid Transporter: From Function to Structure and Role in Cell Biology
    journal, September 2018


    Novel alanine serine cysteine transporter 2 (ASCT2) inhibitors based on sulfonamide and sulfonic acid ester scaffolds
    journal, February 2019


    A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2
    journal, July 2019


      Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.